Method of directed differentiation producing corneal endothelial cells, compositions thereof, and uses thereof
AssigneeAdvanced Cell Technology, Inc.
InventorsKathryn L. McCabe, Shi-Jiang Lu, Robert P. Lanza
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Abstract
This disclosure generally relates to cell-based therapies for treatment of visual disorders, including disorders of the cornea. Methods are exemplified for directed differentiation of corneal cells from stem cells. Compositions of corneal endothelial cells and uses thereof are also provided. Exemplary compositions exhibit improved cell density and/or more “youthful” gene expression relative to cells obtained from donated tissue.
Claims242
A method of producing corneal endothelial cells (CEC), comprising (a) contacting neural crest stem cells (NCSCs) with at least one factor that induces differentiation of said NCSCs into CEC.
The method of any foregoing claim, further comprising culturing said CEC on a matrix.
The method of any foregoing claim, wherein, subsequent to commencement of step (a), said differentiating NCSCs are passaged.
The method of any foregoing claim, wherein the duration of said contacting NCSCs with a factor that induces differentiation of said neural crest cells into CEC is at least 2 days, between 2 and 25 days, or is between 2 and 10 days.
The method of any foregoing claim, wherein step (a) further comprises culturing said NCSCs on a matrix.
The method of any foregoing claim, wherein said NCSCs are obtained from an animal tissue.
The method of any foregoing claim, wherein said NCSCs express one or more markers selected from the group consisting of:
- Sox10, AP2, HNK1, PAX7, p75 (NGFR), and any combination thereof.
The method of any foregoing claim, wherein said CEC express one or more markers selected from the group consisting of:
- Na+/K+ATPase, ZO-1, KLF13, AQP1, Collagen VIII, SLC16A3, CFTR, NBC1, CA2, AE2/SCL4A2, SCL16A1, CA12, CA4, FoxC1, and any combination thereof.
The method of any foregoing claim, wherein said CEC express the markers Collagen VIII, Na+K+ATPase pump, and ZO-1, and do not express the markers vWF and CD31.
The method of any foregoing claim, wherein said CEC express one or more corneal endothelial pump markers.
The method of any foregoing claim, wherein said CEC express one or more periocular neural crest markers.
The method of any foregoing claim, wherein said CEC express one or more cell adhesion and matrix proteins.
The method of any foregoing claim, wherein said CEC express at least one corneal endothelial pump marker, at least one periocular neural crest marker, and at least one cell adhesion and matrix protein.
The method of any foregoing claim, wherein said CEC express the markers Collagen VIII, Na+K+ATPase pump, AQP1, CA2, CA4, CA12, SCL14A2, SLC16A1, SLC16A3, SLC16A7, CFTR, NHE1, ADCY10, PITX2, and FOXC1, and do not express the markers vWF and CD31.
The method of any foregoing claim, wherein said CEC form a monolayer of uniformly sized cells with a predominantly polygonal shape.
The method of any foregoing claim, wherein said CEC form a monolayer of uniformly sized cells with a predominantly hexagonal shape.
The method of any foregoing claim, wherein said CEC allows unidirectional leakage of solutes and nutrients.
The method of any foregoing claim, wherein said CEC exhibit a high level of metabolic activity that is comparable to animal-derived CEC.
The method of any foregoing claim, wherein said CEC are human CEC.
The method of any foregoing claim, wherein said CEC exhibit a culture density of at least 1000 cells/mm2, at least 2000 cells/mm2, between 2000 and 9000 cells/mm2, between 3000 and 8,000 cells/mm2, between 4000 and 7,000 cells/mm2, between 2000 and 4000 cells/mm2, between 2000 and 3500 cells/mm2, or about 2500 cells/mm2.
The method of any foregoing claim, wherein said CEC exhibit a culture density of at least 4000 cells/mm2, at least 5000 cells/mm2, at least 6000 cells/mm2, at least 7000 cells/mm2, at least 8000 cells/mm2, or, at least 9000 cells/mm2 and/or the average size of the CEC in the culture is less than 1/9,000 mm2, less than 1/8,000 mm2, less than 1/7,000 mm2, less than 1/6,000 mm2, less than 1/5,000 mm2, or less than 1/4,000 mm2.
The method of any foregoing claim, wherein said CEC exhibit a statistically significantly decreased level of accumulated oxidative stress and/or DNA damage compared to CEC isolated from a host.
The method of any foregoing claim, further comprising harvesting said CEC as a suspension of cells optionally comprising individual cells and/or clusters or clumps of cells.
The method of any foregoing claim, wherein said CEC comprise at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the cells in the resulting culture.
A composition comprising corneal endothelial cells (CEC) produced according to the method of any foregoing claim.
A composition comprising sheet of CEC having a culture density of at least 1000 cells/mm2, at least 2000 cells/mm2, between 2000 and 9000 cells/mm2, between 3000 and 8,000 cells/mm2, between 4000 and 7,000 cells/mm2, between 2000 and 4000 cells/mm2, between 2000 and 3500 cells/mm2, or about 2500 cells/mm2.
A composition comprising sheet of CEC having a culture density of at least 4000 cells/mm2, at least 5000 cells/mm2, at least 6000 cells/mm2, at least 7000 cells/mm2, at least 8000 cells/mm2, or, at least 9000 cells/mm2 and/or the average size of the CEC in the culture is less than 1/9,000 mm2, less than 1/8,000 mm2, less than 1/7,000 mm2, less than 1/6,000 mm2, less than 1/5,000 mm2, or less than 1/4,000 mm2.
A composition comprising CEC that exhibit a decreased level of accumulated oxidative stress and/or DNA damage compared to CEC isolated from a living host.
A composition comprising corneal endothelial cells (CEC) produced from neural crest stem cells (NCSCs).
A composition comprising corneal endothelial cells (CEC), wherein when transplanted into a human patient or non-human animal said CEC exhibit a statistically significantly improved rate of survival after two years compared to transplantation of donated CEC.
A composition comprising a pharmaceutical preparation comprising a sheet of CEC having a culture density of at least 1000 cells/mm2, at least 2000 cells/mm2, between 2000 and 9000 cells/mm2, between 3000 and 8,000 cells/mm2, between 4000 and 7,000 cells/mm2, between 2000 and 4000 cells/mm2, between 2000 and 3500 cells/mm2, or about 2500 cells/mm2.
A composition comprising a pharmaceutical preparation comprising a sheet of CEC having a culture density of at least 4000 cells/mm2, at least 5000 cells/mm2, at least 6000 cells/mm2, at least 7000 cells/mm2, at least 8000 cells/mm2, or, at least 9000 cells/mm2 and/or the average size of the CEC in the culture is less than 1/9,000 mm2 less than 1/8,000 mm2, less than 1/7,000 mm2, less than 1/6,000 mm2, less than 1/5,000 mm2, or less than 1/4,000 mm2.
A composition comprising a pharmaceutical preparation of CEC that exhibit a decreased level of accumulated oxidative stress and/or DNA damage compared to CEC isolated from a living host.
A composition comprising a pharmaceutical preparation of corneal endothelial cells (CEC) produced from neural crest stem cells (NCSCs).
A composition comprising a pharmaceutical preparation of corneal endothelial cells (CEC), wherein when transplanted into a human patient or non-human animal said CEC exhibit a statistically significantly improved rate of survival after two years compared to transplantation of donated CEC.
A composition comprising a pharmaceutical preparation of corneal endothelial cells (CEC) that differ from CEC derived from a host in the expression of one or more genes.
A composition comprising corneal endothelial cells (CEC) produced from embryonic stem (ES) cells.
A method of producing neural crest stem cells (NCSCs) or NCSC progenitors comprising contacting ES cells with one or more inhibitors of SMA/Mothers Against Decapentaplegic (SMAD) protein signaling.
The method of any one of claims 219 to 229, wherein said second inhibitor of SMAD protein signaling is selected from the group consisting of:
- 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide (SB431542) and derivatives thereof.
Dependencies are inferred from the claim text.